Pseudomonas fluorescens CHA0 Produces Enantio-pyochelin, the Optical Antipode of the Pseudomonas aeruginosa Siderophore Pyochelin
2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês
10.1074/jbc.m707039200
ISSN1083-351X
AutoresZeb A. Youard, Gaëtan L. A. Mislin, Paul Majcherczyk, Isabelle J. Schalk, Cornelia Reimmann,
Tópico(s)Microbial Natural Products and Biosynthesis
ResumoThe siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4 ′R, 2 ″R, 4 ″R (pyochelin I) and 4 ′R, 2 ″S, 4 ″R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed. The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4 ′R, 2 ″R, 4 ″R (pyochelin I) and 4 ′R, 2 ″S, 4 ″R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed. Iron is a cofactor for many redox-dependent enzymes and thus essential for most living organisms including bacteria. But despite its abundance on earth, iron is not freely available to microorganisms under aerobic conditions, as it forms poorly soluble ferric hydroxides in the environment or is tightly bound to transport and storage proteins in mammalian hosts. To acquire iron, bacteria have evolved sophisticated strategies, the most common of which is the production of iron-chelating molecules termed siderophores (1Guerinot M.L. Annu. Rev. Microbiol. 1994; 48: 743-772Crossref PubMed Scopus (529) Google Scholar, 2Wandersman C. Delepelaire P. Annu. Rev. Microbiol. 2004; 58: 611-647Crossref PubMed Scopus (733) Google Scholar). Under iron-limiting growth conditions these molecules are secreted to the environment where they chelate ferric iron and deliver it to the bacterial cytoplasm via specific membrane-associated receptor proteins.The siderophore pyochelin, which is the focus of this study, was isolated in the late 1970s from iron-deficient cultures of Pseudomonas aeruginosa ATCC 15692 (strain PAO1), and its chemical structure was established as 2-(2-o-hydroxyphenyl-2-thiazolin-4-yl)-3-methylthiazolidine-4-carboxylic acid (3Cox C.D. Graham R. J. Bacteriol. 1979; 137: 357-364Crossref PubMed Google Scholar, 4Cox C.D. J. Bacteriol. 1980; 142: 581-587Crossref PubMed Google Scholar, 5Cox C.D. Rinehart K.L. Moore M.L. Cook J.C. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4256-4260Crossref PubMed Scopus (298) Google Scholar, 6Liu P.V. Shokrani F. Infect. Immun. 1978; 22: 878-890Crossref PubMed Google Scholar). Pyochelin forms a 2:1 complex with ferric iron but despite recent physiochemical and crystallographic data, which suggest it to be a tetradentate ligand, the structure of the biologically relevant ferric complex is still unclear (7Cobessi D. Célia H. Pattus F. J. Mol. Biol. 2005; 352: 893-904Crossref PubMed Scopus (107) Google Scholar, 8Schlegel K. Lex J. Taraz K. Budzikiewicz H. Z. Naturforsch. 2006; 61: 263-266Crossref PubMed Scopus (18) Google Scholar, 9Tseng C.F. Burger A. Mislin G.L.A. Schalk I.J. Yu S.S.-F. Chan S.I. Abdallah M.A. J. Biol. Inorg. Chem. 2006; 11: 419-432Crossref PubMed Scopus (44) Google Scholar). Pyochelin has three chiral centers at the C4′, C2″, and C4″ positions and is extracted from P. aeruginosa PAO1 as a mixture of two inter-convertible diastereoisomers whose absolute configuration was determined as 4′R, 2″R, 4″R (pyochelin I) and 4′R, 2″S, 4″R (pyochelin II) (Fig. 1 and Refs. 10Ankenbauer R.G. Toyokuni T. Staley A. Rinehart K.L. Cox C.D. J. Bacteriol. 1988; 170: 5344-5351Crossref PubMed Google Scholar, 11Rinehart K.L. Staley A.L. Wilson S.R. Ankenbauer R.G. Cox C.D. J. Org. Chem. 1995; 60: 2786-2791Crossref Scopus (64) Google Scholar). The presence of iron(III) and zinc(II) were shown to induce a shift from pyochelin II to pyochelin I by converting the S configuration to the R configuration at the chiral center C2″ (12Ino A. Murabayashi A. Tetrahedron. 2001; 57: 1897-1902Crossref Scopus (41) Google Scholar, 13Schlegel K. Taraz K. Budzikiewicz H. BioMetals. 2004; 17: 409-414Crossref PubMed Scopus (35) Google Scholar). Similar metal-induced shifts may occur in the other diastereoisomer pairs (Fig. 1).Pyochelin is a condensation product of salicylate and two cysteinyl residues, its biosynthesis in P. aeruginosa requires proteins encoded by the two divergent operons pchDCBA and pchEFGHI (14Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (101) Google Scholar, 15Reimmann C. 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Bacteriol. 2001; 183: 813-820Crossref PubMed Scopus (87) Google Scholar, 20Patel H.M. Walsh C.T. Biochemistry. 2001; 40: 9023-9031Crossref PubMed Scopus (93) Google Scholar, 21Quadri L.E.N. Keating T.A. Patel H.M. Walsh C.T. Biochemistry. 1999; 38: 14941-14954Crossref PubMed Scopus (114) Google Scholar). Specialized tailoring domains in PchE (E domain) and PchF (MT domain) are responsible for the epimerization of the L-cysteinyl to the D-cysteinyl residue during formation of the thiazoline ring (22Patel H.M. Tao J. Walsh C.T. Biochemistry. 2003; 42: 10514-10527Crossref PubMed Scopus (44) Google Scholar) and for methylation of the nitrogen in the thiazolidine ring (20Patel H.M. Walsh C.T. Biochemistry. 2001; 40: 9023-9031Crossref PubMed Scopus (93) Google Scholar), respectively. The expression of the pyochelin biosynthetic genes is strongly induced by pyochelin (14Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (101) Google Scholar) and depends on the AraC-type regulator PchR (23Heinrichs D.E. Poole K. J. Bacteriol. 1993; 175: 5882-5889Crossref PubMed Google Scholar, 24Michel L. González N. Jagdeep S. Nguyen-Ngoc T. Reimmann C. Mol. Microbiol. 2005; 58: 495-509Crossref PubMed Scopus (91) Google Scholar).Pyochelin has also been isolated from other pseudomonads and closely related bacteria (25Budzikiewicz, H. (2003) in Progress in the Chemistry of Organic Natural Products (Herz, W., Falk, H., and Kirby, G. W., eds), Vol. 87, Springer, WienGoogle Scholar, 26Castignetti D. Curr. Microbiol. 1997; 34: 250-257Crossref PubMed Scopus (21) Google Scholar, 27Darling P. Chan M. Cox A.D. Sokol P.A. Infect. Immun. 1998; 66: 874-877Crossref PubMed Google Scholar, 28Phoebe C.H. Combie J. Albert F.G. Van Tran K. Cabrera J. Correira H.J. Guo Y. Lindermuth J. Rauert N. Galbraith W. Selitrennikoff C.P. J. Antibiot. 2001; 54: 56-65Crossref PubMed Scopus (49) Google Scholar, 29Sokol P. J. Clin. Microbiol. 1986; 23: 560-562Crossref PubMed Google Scholar) although the configuration of the three chiral centers was not always determined. Interestingly, we discovered that the PchE peptide synthetase of the sequenced Pseudomonas fluorescens strain Pf-5 (30Paulsen I.T. Press C.M. Ravel J. Kobayashi D.Y. Myers G.S.A. et al.Nat. Biotechnol. 2005; 23: 873-878Crossref PubMed Scopus (496) Google Scholar) lacks the epimerase domain required to generate the R configuration at the asymmetric center C4′, which prompted us to analyze the pyochelin cluster of the closely related P. fluorescens strain CHA0 and to elucidate the structure of the molecule that it specified. Here we report that CHA0 and Pf-5 synthesize the enantiomer of pyochelin for which we propose the name enantio-pyochelin (Fig. 1). We show that enantio-pyochelin promotes growth under iron-limiting conditions and induces the expression of its biosynthetic genes in CHA0, but is not recognized as a siderophore and signaling molecule by P. aeruginosa.EXPERIMENTAL PROCEDURESChemicals—Metal-free silica was obtained by gentle stirring of Merck Geduran Kieselgel Si 60 (40–63 μm) with 1 n HCl at 25 °C for 12 h. The resulting suspension was filtered and washed several times with MilliQ water until the pH of the filtrate was between 5 and 6. The silica was then dried, first under reduced pressure and afterward in an oven at 110 °C for 48 h.Media and Growth Conditions—Bacteria were routinely grown on nutrient agar and in nutrient yeast broth (31Stanisich V.A. Holloway B.W. Genet. Res. Camb. 1972; 19: 91-108Crossref PubMed Scopus (97) Google Scholar) at 37 °C (P. aeruginosa and Escherichia coli) or 30 °C(P. fluorescens). For (enantio-) pyochelin production and green fluorescent protein (GFP) 2The abbreviation used is: GFP, green fluorescent protein. reporter assays, strains were cultivated in the complex medium GGP (32Carmi R. Carmeli S. Levy E. Gough F.J. J. Nat. Prod. 1994; 57: 1200-1205Crossref PubMed Scopus (50) Google Scholar) in which iron is present but not immediately accessible (probably because it is bound to proteins and peptides), thus inducing the expression of (enantio-) pyochelin biosynthesis and uptake genes. As siderophore-negative mutants grow well in GGP, the minimal medium M9 (33Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar) with 0.5% glycerol as a carbon source was used for siderophore utilization assays. Iron limitation was achieved in this medium by adding the iron chelator 2, 2′-dipyridyl at 500 μm. Antibiotics were added to the growth media at the following concentrations: tetracycline 25 μgml–1 for E. coli and 125 μgml–1 for Pseudomonas. To counterselect E. coli donor cells in gene replacement experiments, chloramphenicol was used at a concentration of 10 μgml–1; mutant enrichment was performed with tetracycline at a final concentration of 20 μgml–1 and carbenicillin (for P. aeruginosa) or cycloserine (for P. fluorescens) at final concentrations of 2 mg ml–1 and 50 mg ml–1, respectively.DNA Manipulation and Sequencing—Small- and large-scale preparations of plasmid DNA were made with the QIAprep Spin Miniprep kit (Qiagen, Inc.) and Jetstar kit (Genomed GmbH), respectively. DNA fragments were purified from agarose gels with the Geneclean II kit (Bio 101, La Jolla, CA) or the MinElute and QIAquick Gel Extraction kits from Qiagen (Qiagen, Inc.). DNA manipulations were performed according to standard procedures (33Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar). Transformation of E. coli, P. aeruginosa, and P. fluorescens was carried out by electroporation (34Farinha M.A. Kropinski A.M. FEMS Microbiol. Lett. 1990; 58: 221-225PubMed Google Scholar). All constructs involving PCR techniques were verified by sequence analysis. Sequencing was performed with the BigDye Terminator Cycle Sequencing Kit and an ABI-PRISM 373 automatic sequencer (Applied Biosystems) or was carried out commercially. The DNA sequence of the P. fluorescens CHA0 pchR-pchDHIEFKCBA genomic region was deposited at GenBank™ under accession number EU088199. Sequences were compared with those from P. aeruginosa PAO1 and P. fluorescens Pf-5. Data base searches were conducted at NCBI using BLAST algorithms.Mutant Construction—Deletion of the enantio-pyochelin biosynthesis operon pchDHIEFKCBA in P. fluorescens was achieved by gene replacement as described previously (35Laville J. Blumer C. von Schroetter C. Gaia V. Défago G. Haas D. J. Bacteriol. 1998; 180: 3187-3196Crossref PubMed Google Scholar, 36Schnider U. Keel C. Blumer C. Troxler J. Défago G. Haas D. J. Bacteriol. 1995; 177: 5387-5392Crossref PubMed Google Scholar). The suicide plasmid pME7535 was constructed as follows. Two PCR fragments were generated from chromosomal DNA of P. fluorescens CHA0 (37Voisard, C., Bull, C., Keel, C., Laville, J., Maurhofer, M., Schnider, U., Défago, G., and Haas, D. (1994) in Molecular Ecology of Rhizosphere Microorganisms (O'Gara, F., Dowling, D., and Boesten, eds), pp. 67–89, VCH Publishers, Weinheim, GermanyGoogle Scholar) using the primers pchD-1 (ACGTGGTACCATGTCCACTTTCGATGACC) together with primer pchD-3 (ACGTGGATCCGGTGGAGCCGCCGGAGGC) and pchA-1 (ACGTGGATCCGGCACCCTCAACACCGTGG) together with primer pchA-2 (ACGTAAGCTTCTACAGGGAGAGGCCGAGC). Fragment 1, cleaved with KpnI and BamHI, was ligated to BamHI- and HindIII-trimmed fragment 2 and cloned into the suicide vector pME3087 (37Voisard, C., Bull, C., Keel, C., Laville, J., Maurhofer, M., Schnider, U., Défago, G., and Haas, D. (1994) in Molecular Ecology of Rhizosphere Microorganisms (O'Gara, F., Dowling, D., and Boesten, eds), pp. 67–89, VCH Publishers, Weinheim, GermanyGoogle Scholar) between the KpnI and HindIII sites. Plasmid pME7535 was then introduced into the wild-type strain CHA0 and its pyoverdine-negative derivative CHA400 (38Keel C. Voisard C. Berling C.H. Kahr G. Défago G. Phytopathology. 1989; 79: 584-589Crossref Google Scholar) to generate the corresponding mutants CHA1084 and CHA1085, respectively.The pyoverdine and pyochelin-negative P. aeruginosa mutant PAO6399 was constructed with the previously described suicide plasmid pME7152 (39Michel L. Bachelard A. Reimmann C. Microbiology. 2007; 153: 1508-1518Crossref PubMed Scopus (49) Google Scholar), which was mobilized from E. coli S17-1 (40Simon R. Priefer U. Pühler A. Bio-technology. 1983; 1: 784-790Crossref Scopus (5593) Google Scholar) to the pchE deletion mutant PAO6310 (14Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (101) Google Scholar) and chromosomally integrated with selection for tetracycline resistance. Excision of the vector was obtained by enrichment for tetracycline-sensitive cells (41Ye R.W. Haas D. Ka J.O. Krishnapillai V. Zimmermann A. Baird C. Tiedje J.M. J. Bacteriol. 1995; 177: 3606-3609Crossref PubMed Google Scholar). All gene replacement mutants were checked by PCR. The absence of siderophore production by CHA1085 and PAO6399 was verified on CAS agar (42Schwyn B. Neilands J.B. Anal. Biochem. 1987; 160: 47-56Crossref PubMed Scopus (4333) Google Scholar).Construction of GFP Reporters—Translational fusions of pchD (CHA0) and pchE (PAO) to the gfp gene carried by the vector pPROBE-TT′ (43Miller W.G. Leveau J.H.J. Lindow S.E. Mol. Plant-Microbe Interact. 2000; 13: 1243-1250Crossref PubMed Scopus (436) Google Scholar) were constructed by overlap extension PCR as follows. A 0.4-kb PCR fragment 1 was amplified from CHA0 chromosomal DNA using the primers PB1 (ACGTGAATTCATGGCGAACTCCCTGTGG) and PB9 (GCTGTCTCCTGATGTTTTTTACG) and a 0.23-kb PCR fragment 2 was amplified from pPROBE-TT′ using the primers PB11 (AAAAAACATCAGGAGACAGCATGAGTAAAGGAGAAGAAC) and PB4 (GCCGTTTCATATGATCTGGG). Fragments 1 and 2, which are complementary to each other over a length of 20 nucleotides, were mixed in equimolar amounts, elongated during 10 PCR cycles with Taq polymerase and dNTPs and subsequently amplified during 20 PCR cycles with primers PB1 and PB4. The resulting PCR fragment C was cleaved with EcoRI and NdeI and ligated to pPROBE-TT′ cut with the same enzymes. This yielded the pchDCHA0–gfp reporter construct pME7585. The pchEPAO–gfp reporter plasmid pME7588 was constructed in a similar way. PCR fragment 1 (0.34 kb) was amplified from chromosomal DNA of PAO1 using the primers pchEgfp1 (ACGTGAATTCCTGCAGGAATACCGCCTG) and pchEgfp2 (GGGGGCTCCCTAGGGCAAGC) and PCR fragment 2 (0.23 kb) was amplified from pPROBE-TT′ with primers pchEgfp3 (GCTTGCCCTAGGGAGCCCCCATGAGTAAAGGAGAAGAAC) and PB4. Subsequent overlap extension PCR yielded a 0.57-kb fragment, which was cleaved with EcoRI and NdeI and cloned into pPROBE-TT′.Extraction and Purification of (Enantio-) pyochelin—Enantio-pyochelin was extracted and purified from CHA400 as follows. The cell-free supernatant from a 2-day culture grown in GGP medium was acidified to pH 1–2 and extracted with 1 volume ethyl acetate. For thin-layer chromatography, mass spectrometry, and polarimetric measurements, this extract was washed successively with 0.5 n HCl and MilliQ water. The organic phase was dried over Na2SO4, and filtered. Solvents were evaporated under reduced pressure. The resulting orange oily residue was purified by chromatography on a metal-free silica gel column eluted with a 90:10 CH2Cl2/acetone mixture. Enantio-pyochelin was obtained as a pale yellow powder after evaporation of solvents under reduced pressure.For use in NMR experiments, chiral HPLC, and bioassays, the crude ethyl acetate extract was dried by evaporation, dissolved in methanol and further purified by HPLC using a VP 250/10 Nucleosil 100-7 C18 preparative column (Macherey-Nagel) and a Waters™ HPLC system equipped with a 2487 Dual λ Absorbance Detector. Aliquots (300 μl) were injected into the HPLC system and separated at room temperature using an isocratic gradient consisting of 70% Solvent A (H2O + 0.1% trifluoroacetic acid) and 30% Solvent B (95% acetonitrile + 0.1% trifluoroacetic acid) and a flow rate of 1 ml/min. Two peaks with maximal absorbance at 235 and 260 nm (absorbance of the thiazoline ring) were collected and dried by evaporation. These peaks with retention times of 16 and 24 min, correspond to the two diastereoisomers of enantio-pyochelin as determined by NMR, chiral HPLC, and bioassays (see "Results"). Extraction and purification of pyochelin from P. aeruginosa was done in a similar way and has been previously described (14Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (101) Google Scholar).GFP Reporter Assays—Pseudomonas strains carrying translational gfp fusions were grown in 96-well black microtiter plates (Greiner bio-one) with a flat transparent bottom. Each well contained 200 μl of GGP medium and was inoculated with 3 μl of a bacterial preculture grown overnight in the same medium. HPLC-purified pyochelin or enantio-pyochelin was dissolved in methanol to a concentration of 10 mm (determined by the CAS assay (42Schwyn B. Neilands J.B. Anal. Biochem. 1987; 160: 47-56Crossref PubMed Scopus (4333) Google Scholar)) and added to the growth medium at the concentrations indicated. Microtiter plates were incubated at 37 °C (P. aeruginosa) or 30 °C(P. fluorescens) with orbital shaking at 500 rpm. At each given time point growth (A600) and green fluorescence (excitation at 480 nm and emission at 520 nm) were measured from triplicate cultures using a Fluostar fluorescence microplate reader (BMG Lab Technologies). For each individual measurement the green fluorescence value was divided by the respective A600 value giving the specific fluorescence of cells expressed as relative fluorescence units. The green fluorescence of cells carrying the empty vector pPROBE-TT′ was determined for background fluorescence correction.Siderophore-mediated Growth Promotion—Utilization of pyochelin or enantio-pyochelin as a siderophore was measured with liquid growth assays. Microtiter wells containing 200 μlof M9-glycerol minimal medium with or without the iron chelator 2, 2′-dipyridyl at 500 μm were inoculated with 3 μl of precultures grown overnight in M9-glycerol medium. HPLC-purified pyochelin or enantio-pyochelin was added to the growth medium at 20 μm. Growth (A600) at 500 rpm was recorded by a microplate reader (BMG Lab Technologies).Thin-layer Chromatography (TLC)—Analytical TLC was performed with Merck TLC silica gel 60F254 on aluminum sheets using n-butyl alcohol/water/acetic acid 4:1:1 (v/v/v) as the mobile phase. Compounds were detected either by fluorescence at 365 nm or by spraying the TLC sheet with a solution of FeCl3 in MeOH. Pictures were recorded with a Kodak DX7590 digital camera and processed using the Kodak EasyShare Picture Editor.Mass Spectrometry—Electrospray mass spectrometry experiments were performed on a microTOF LC from Brucker Daltonics.NMR Experiments—NMR experiments (NOESY, COSY, ROESY) were performed at 300, 400, or 500 MHz using Brucker Advance spectrometers on solutions of pyochelin, enantio-pyochelin, or neopyochelin in deuterated acetone.Polarimetry—Pyochelin and enantio-pyochelin samples were dissolved in acetone at a concentration of 10 mg ml–1 and optical activities were determined at 20 °C in a Hellma glass cell (100-mm length) using a Perkin Elmer Model 341 polarimeter.Separation of Pyochelin and Enantio-pyochelin by Chiral HPLC—Samples (10 μg in 20 μl methanol) of pyochelin or enantio-pyochelin were injected into a Merck Hitachi HPLC system equipped with a L-7450A Diode Array Detector, and separated on a Daicel Chiralcel® OD-H analytical column at room temperature using an isocratic gradient consisting of 95% Solvent A (heptane + 0.1% trifluoroacetic acid) and 5% Solvent B (ethanol + 0.1% trifluoroacetic acid) and a flow rate of 1 ml/min. Pyochelin and enantio-pyochelin were detected by their absorption at 254 nm.Synthesis and Purification of Pyochelin, Neopyochelin, and Enantio-pyochelin—Pyochelin and neopyochelin were synthesized using protocols published previously (44Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 249-256Crossref Scopus (61) Google Scholar, 45Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 9397Crossref Google Scholar). The synthesis of enantio-pyochelin was inspired by the same protocol and started with the condensation of 2-hydroxybenzonitrile with l-cysteine in a buffered hydromethanolic medium (46Bergeron R.J. Wiegand J. Dionis J.B. Egli-Kamarkka M. Frei J. Huxley-Tencer A. Peter H. J. Med. Chem. 1991; 34: 2072-2078Crossref PubMed Scopus (109) Google Scholar). The resulting thiazoline, recrystallized from a mixture of ethanol and n-hexane, was then converted into the corresponding Weinreb amide, and the latter was reduced to an aldehyde with lithium aluminum hydride (44Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 249-256Crossref Scopus (61) Google Scholar, 45Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 9397Crossref Google Scholar). The aldehyde was condensed with N-methyl-d-cysteine prepared from d-cysteine according to the procedure of Blondeau et al. (47Blondeau P. Berse C. Gravel D. Can. J. Chem. 1967; 45: 49-52Crossref Google Scholar). At this stage, the synthesis of enantio-pyochelin (and also that of pyochelin and neopyochelin), leads to a mixture of four stereoisomers (11Rinehart K.L. Staley A.L. Wilson S.R. Ankenbauer R.G. Cox C.D. J. Org. Chem. 1995; 60: 2786-2791Crossref Scopus (64) Google Scholar, 44Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 249-256Crossref Scopus (61) Google Scholar, 45Zamri A. Abdallah M.A. Tetrahedron. 2000; 56: 9397Crossref Google Scholar). Among them, the two naturally occuring diastereoisomers of pyochelin or enantio-pyochelin were separated from the two "neo" isomers by chromatography on a metal-free silica gel column eluted with a gradient of acetone in CH2Cl2 (from 5:95 to 30:70). The resulting yellow oily residue was converted into a pale yellow powder by precipitation in a mixture of acetone and n-hexane followed by evaporation under reduced pressure.RESULTSComparison of the Pyochelin Biosynthetic Genes from P. aeruginosa with Their Counterparts in P. fluorescens—Putative pyochelin biosynthetic (pch) genes have been located in the recently sequenced genome of P. fluorescens strain Pf-5 (30Paulsen I.T. Press C.M. Ravel J. Kobayashi D.Y. Myers G.S.A. et al.Nat. Biotechnol. 2005; 23: 873-878Crossref PubMed Scopus (496) Google Scholar). We sequenced the corresponding genes from the closely related strain CHA0. Comparison of the deduced amino acid sequences revealed identities of 98 to 99% between the pch genes in the two strains (not shown). When these genes were compared with the well-characterized pch genes of P. aeruginosa PAO1 (14Reimmann C. Serino L. Beyeler M. Haas D. Microbiology. 1998; 144: 3135-3148Crossref PubMed Scopus (101) Google Scholar, 15Reimmann C. Patel H.M. Serino L. Barone M. Walsh C.T. Haas D. J. Bacteriol. 2001; 183: 813-820Crossref PubMed Scopus (87) Google Scholar, 16Serino L. Reimmann C. Baur H. Beyeler M. Visca P. Haas D. Mol. Gen. Genet. 1995; 249: 217-228Crossref PubMed Scopus (146) Google Scholar, 17Serino L. Reimmann C. Visca P. Beyeler M. Della Chiesa V. Haas D. J. Bacteriol. 1997; 179: 248-257Crossref PubMed Google Scholar), three major differences were discovered. Firstly, the genomic organization of the pch genes is different (Fig. 2). In PAO1 the pch genes are organized in two operons flanking the regulatory gene pchR. In Pf-5 and CHA0 all pch genes seem to be contained within one operon. Second, an epimerization domain is absent from PchEPf-5/CHA0. Third, there is no homology between PchGPAO1 and its presumed counter-part in Pf-5 and CHA0 (named here PchK, see Fig. 2). A motif search indicated that PchK could have a reductase and/or epimerase function.FIGURE 2Organization of the pyochelin genes in P. aeruginosa and their counterparts in P. fluorescens Pf-5 and CHA0. The physical maps are based on total genome sequences of P. aeruginosa PAO1 (62Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. et al.Nature. 2000; 406: 959-964Crossref PubMed Scopus (3354) Google Scholar) and P. fluorescens Pf-5 (30Paulsen I.T. Press C.M. Ravel J. Kobayashi D.Y. Myers G.S.A. et al.Nat. Biotechnol. 2005; 23: 873-878Crossref PubMed Scopus (496) Google Scholar). The sequence of the pchR-pchDHIEFKCBA region in CHA0 is available at GenBank™ under accession number EU088199. Deduced amino acid sequences of P. aeruginosa and P. fluorescens pch genes (gray) are between 35 and 60% identical except for pchG (white) and its presumed counterpart pchK (black), which are not related. Note that pchE is smaller in the P. fluorescens strains than in P. aeruginosa PAO1 due to the absence of an epimerase coding region (boxed in pchEPAO).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Biological Activity of the P. fluorescens Siderophore—To characterize the molecule specified by the pchDHIEFKCBA operon, a culture supernatant of the pyoverdine-negative strain CHA400 was extracted with ethyl acetate and the siderophore was purified by HPLC as described under "Experimental Procedures." We first tested its biological activity as a siderophore in growth promotion assays. As shown in Fig. 3, unmodified M9-glycerol medium contained sufficient iron to support growth of the siderophore-negative mutants CHA1085 (Fig. 3A) and PAO6399 (Fig. 3B) but when the medium was amended with the iron chelator 2, 2′-dipyridyl, these mutants were no longer able to grow. Growth of CHA1085 was restored by the addition of 20 μm siderophore purified from P. fluorescens CHA400 whereas equal amounts of HPLC-purified pyochelin from PAO1 had no effect, indicating that pyochelin cannot be used as a siderophore by P. fluorescens (Fig. 3A). Similar results were obtained in experiments performed with P. aeruginosa (Fig. 3B) where the endogenous siderophore pyochelin was able to promote growth of the pyoverdine- and pyochelin-negative mutant PAO6399, while no growth promotion was observed under iron limitation with the P. fluorescens siderophore.FIGURE 3Siderophore-dependent growth promotion under iron limitation. Siderophore-negative mutants of P. fluorescens (CHA1085; A) or P. aeruginosa (PAO6399; B) were grown in unmodified M9-glycerol medium (▪) or in M9-glycerol medium containing the iron chelator 2, 2′-dipyridyl (▴) and 20 μm HPLC-purified siderophore extracted from CHA400 (⋄) or PAO1 (i.e. pyochelin; ○). Growth was assessed over a period of 100 h. A600 values represent the means ± standard deviations from three parallel cultures.View Large
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